Caveolin-2 (Cav-2) knockout (KO) mice develop hyperproliferative phenotype in the lung with microvascular endothelial cells (ECs) as the likely major hyperproliferating cell type. This suggests that Cav-2 regulates EC proliferation, differentiation and physiological angiogenesis in the lung. Also, Cav-2 KO mice display enhanced growth factor-induced angiogenesis, suggesting the role for Cav-2 in regulating postnatal angiogenesis involving systemic microvasculature. Our new data with recently isolated mouse lung ECs (MLECs) from Cav-2 KO vs. WT mice suggest that Cav-2 is directly involved in regulating EC proliferation and differentiation in vitro. We have also determined that Cav-2 is phosphorylated at two serine residues, 23 and 36, in human ECs and that serine phosphorylation of Cav-2 is regulated in mitotic ECs. Cav-2 can also be phosphorylated at tyrosines 19 and 27. Thus, N-terminal serine and tyrosine phosphorylation of Cav-2 is a likely molecular mechanism by which Cav-2 regulates EC proliferation and differentiation. We hypothesize that cav-2 and its phosphorylation negatively regulates EC proliferation, differentiation, and postnatal angiogenesis. We will: 1. Examine the role of Cav-2 and the phosphorylation of its serine 23 and 36 and tyrosine 19 and 27 in regulating EC proliferation;2. Examine the role of Cav-2 and its phosphorylation in regulating EC differentiation/morphogenesis into capillary-like structures in vitro;3A. Examine the role of Cav-2 in regulating physiological angiogenesis involving lung microvascular ECs during postnatal growth;3B.Examine the role of Cav-2 expressed in host microvasculature in pathological, tumor-induced angiogenesis upon subcutaneous implantation of tumor cells. In Aims 1 and 2, we will use a combination of Cav-2 KO vs. WT MLECs and human ECs treated with Cav-2 vs. control sh/siRNAs. We will also, use adenoviral and regulated lentiviral expression systems to re-express WT and phosphorylation mutants of Cav-2 in Cav-2 KO MLECs. In Aim 3, we will perform direct comparisons of physiological (A) and pathological (B) angiogenesis in Cav-2 KO vs. WT littermate mice. Collectively, these three aims will provide important mechanistic information as to the role of Cav-2 in regulating EC proliferation, differentiation as well as physiological and pathological angiogenesis in vivo. PUBLIC HEALTH RELEVANCE: The overall goal of this research proposal is to understand the role of the membrane protein called caveolin-2 in regulating endothelial cell proliferation, differentiation and their combined contribution to angiogenesis. Angiogenesis is the process of new blood vessel formation, which is essential for physiological growth, development, wound healing, as well as pathological tumor growth. Thus, understanding the molecular mechanisms controlling angiogenesis is of key significance. The knowledge gained from this research will facilitate the treatment of cardiovascular and pulmonary disease, injuries, and cancer.